Plasmacytoid dendritic cells (pDCs) are principal type I interferon(IFN)-producing cells in bone marrow and lymphoid organs that are thought to play critical roles in anti-viral immunity. Upon activation through Toll-like receptors (TLRs), pDCs secrete IFN and mature to an antigen-presenting state in which they are able to elicit adaptive immune responses including CD4+ T cell polarization. The pDC developmental pathway proceeds through a common DC progenitor (CDP), which generates both pDCs and conventional DCs. PDC production depends on signaling by the receptor tyrosine kinase Flt3 and the transcription factors STAT3 and E2-2. A pDC subset was identified within Peyer's Patches (PP) in the gut that is unable to produce type I IFNs upon TLR triggering. This subset is implicated in oral tolerance; however the developmental origin and role for PP pDCs in eliciting adaptive immune responses are largely unknown. We found that PP pDC accrual required cell autonomous signaling via type I IFNs and STAT1, suggesting a distinct developmental or conditioning pathway compared to BM or splenic pDCs. Furthermore, we found that PP pDCs correlate with IL-17-secreting CD4+ T (Th17) cells in PPs. We hypothesize that PP pDCs arise from the same developmental pathway as pDCs localized in BM and spleen (i.e., via CDPs in response to Flt3-STAT3 signaling), however conditioning within the gut microenvironment may render distinct functional attributes including the ability to induce Th17 cell generation. We will test this hypothesis by investigating the pathways that regulate the accrual of pDCs within PPs (Aim 1). We will assess whether purified BM progenitors such as CDPs reconstitute PP pDCs in lethally irradiated mice or in a novel transplant model using Ifnar-/- animals that lack PP pDCs. Molecular pathways required for PP pDC generation will be examined by transplant experiments with BM progenitors from Stat1-/-, STAT3-deficient, Ifnar-/- or Tcf4-/- (E2-2-deficient) mice in steady state or in response to cytokines that elicit BM and spleen pDC generation (i.e., Flt3L, IFN-1). In Aim 2, we will determine whether PP pDCs are functionally distinct from BM or splenic pDCs in terms of eliciting CD4+ T cell responses, using in vitro CD4+ T cell polarization assays with purified PP, BM and splenic pDCs and naove CD4+ T cells. In addition, CD4+ T cell populations within PPs will be correlated with PP pDC reconstitution in Aim 1 to further examine the relationship between PP pDCs and Th cell responses, and the activation profiles of PP pDCs will be compared to BM and spleen pDCs by measuring candidate cytokine production, activating and inhibitory molecule expression and transcriptional profiles. Successful completion of this project will provide a mechanistic view of the PP pDC developmental pathway, and thus may contribute to development of therapeutic applications that enable redirection of the immune response in diseases associated with gastrointestinal inflammation. PUBLIC HEALTH RELEVANCE: The goal of this project is to discover pathways that control the generation and function of a specialized population of dendritic cells within the intestinal tract, which are required for maintaining a balanced response to foreign materials. Knowledge from this project may aid in the future development of therapies to suppress or redirect gastrointestinal inflammation during immune disease or cancer.